This invention relates to a composition comprising a purified mycobacterial lipid cell-wall component or log or derivative thereof and a pharmaceutically acceptable excipient, medium, carrier or adjuvant and the use of the purified mycobacterial lipid cell-wall component or analog or derivative thereof and the composition containing it in the prevention, treatment and diagnosis of disease.
It has been known for a long time that BCG*) vaccination leads to the induction of a positive tuberculin skin test, resulting in delayed type hypersensitivity (DTH). This delayed hypersensitivity in turn has been considered to be indicative of the successful induction of protective immunity against tuberculosis and has led to the almost world-wide BCG immunization in the 1950s-1970s. This convenient test is in fact the only immunological criterion/parameter on which epidemiological assessments of the effectiveness of the immunization have been based.
BCG: (Bacillus of Calmette and Guerin) Calmette and Guerin attenuated a strain of M. bovis by passaging it 231 times over a period of 13 years through a medium containing glycerine and ox-bile. 
This view is no longer generally accepted and many immunologists are of the opinion that
i) the induction of DTH is not directly related to the degree of protective immunity;
ii) the protective efficacy obtained in vaccination with BCG varies between 0 to 80% (Snider, 1994) and in addition
iii) BCG vaccination has detrimental side-effects being partially responsible for tissue destruction in patients, without offering sufficient protection (Fine, 1994).
The unsatisfactory results observed and reported in a number of countries with the BCG vaccine currently used for the prevention of the spread of tuberculosis (Dolin, Raviglione and Koch, 1994; Snider, 1994) could be explained by:
i) variations between BCG vaccines, which could be caused by strain variation or by differences between manufacturing processes;
ii) differences in pathogenesis of Mycobacterium tuberculosis; 
iii) differences in the exposure to the environmental mycobacteria. The environmental mycobacteria may act antagonistically or synergistically with BCG;
iv) genetic differences between population groups subjected to vaccination with BCG;
v) differences in nutrition and exposure to sunlight between various population groups;
vi) differences between designs of various studies;
vii) inadequacies of the criteria used for the evaluation of protective effects of vaccination with BCG.
Efforts directed at finding an effective vaccine capable of inducing long-lasting immunity have centered over the last decade on three main approaches:
i) identifying xe2x80x9cprotectivexe2x80x9d antigens and epitopes of M. tuberculosis presented by macrophages and recognized by human lymphocytes;
ii) developing a DNA-based vaccine with protective antigen and interleukin genes (Lowrie et al., 1994);
iii) identifying which types of cells of the immune system and which types of cytokines are involved in tuberculosis in order to manipulate their activity towards offering a cure or protection against tuberculosis.
According to one aspect of the invention there is provided a conjugate comprising an organic carrier and a purified lipid cell-wall component associated therewith, provided that if the organic carrier is a protein, it is not bovine serum albumin (BSA), gelatin, keyhole limpets haemocyanin or the CD1 molecule.
The organic carrier may be a protein excluding bovine serum albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1 molecule.
The protein may be a microbial protein. More specifically, it may be a modified bacterial protein and may be derived from a bacterium from the genus Mycobacterium, Corynebacterium, Nocardia or Rhodococcus. It may be a heat-shock protein, such as heat-shock protein 60 (HSP60) or heat-shock protein 65 (HSP65), or it may be a serum protein from an animal. The animal may have a mycobacterial infection, such as a Mycobacterium tuberculosis infection. The animal may be a mammal, typically a human.
Alternatively, the protein may be derived from a mammal, particularly a human, and is preferably a protein which mimics the structure of collagen or a collagen-derived protein or a plasma protein, such as the collagen-like segment of human serum component C1q.
Alternatively, the carrier may be a carbohydrate such as galactomannan or arabinogalactan or a lipopolysaccharide.
Further alternatively, the organic carrier may be a micelle, such as a liposome.
According to another aspect of the invention there is provided a diagnostic kit comprising a support containing a conjugate as described above immobilised thereon.
According to another aspect of the invention there is provided a pharmaceutical composition which comprises a therapeutic, prophylactic or tolerogenic amount of a conjugate as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component or analog or derivative thereof associated therewith or a biologically active purified lipid cell-wall component or analog or derivative thereof and a pharmaceutically acceptable or compatible pharmaceutical excipient, medium, carrier or adjuvant.
The organic carrier may be a protein including bovine serum albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1 molecule, or may be a carbohydrate or may be a micelle.
The pharmaceutical composition may also contain at least one immunomodulator. The immunomodulator may be a cytokine. The cytokine may be an interleukin, such as interleukin 4 (IL4), interleukin 10 (IL10) or interleukin 12 (IL12), or may be an interferon.
The suitable pharmaceutical carrier or adjuvant which may also be suitable for veterinary applications may be a solid, such as polymer dust, a liquid, such as an oil, typically Marcol 52, or a water-in-oil emulsion, typically Freund""s Incomplete Adjuvant (FIA), or a solution, typically a saline solution or PBS, in which case the composition may be in the form of a suspension or a vapourised liquid, typically a neubulisable physiological saline solution, or a gas, or a transdermal delivery system.
The composition may comprise a therapeutic, prophylactic or tolerogenic amount of the purified lipid cell-wall component.
The pharmaceutical composition may comprise about 5 xcexcg or less, typically 1 xcexcg, of the purified lipid cell-wall component per ml of the composition.
A unit dose of the pharmaceutical composition for administration to a human subject preferably comprises from about 5 to 10 mg of the purified lipid cell-wall component.
According to another aspect of the invention there is provided a vaccine containing a purified lipid cell-wall component or analog or derivative thereof or a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith for use in preventing an immune disorder or an inflammatory condition in a subject.
According to another aspect of the invention there is provided a vaccine containing a purified lipid cell-wall component or analog or derivative thereof or a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier, except bovine serum albumin (BSA) and a purified lipid cell-wall component associated therewith, for use in preventing a microbial infection in a subject.
The organic carrier may be a protein including bovine serum albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1 molecule, or may be a carbohydrate or may be a micelle.
According to another aspect of the invention there is provided an isolated antibody which is capable of forming, separately, an antigen/antibody complex with any two or more of the following antigens: a purified lipid cell-wall component derived from a microorganism: a protein derived from a bacterial species or from a mammal; a conjugate as described above; and a conjugate comprising any organic carrier and a purified mycobacterial lipid cell-wall component associated therewith.
The organic carrier may be a protein including bovine serum albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1 molecule, or may be a carbohydrate or may be a micelle.
According to another aspect of the invention there is provided a method of diagnosing a microbial infection in a subject comprising the step of contacting a sample from the subject with a conjugate as described above or with a support containing a conjugate as described above; and detecting any reaction between the conjugate and the sample.
More preferably, the method of diagnosis may comprise the step of detecting the binding of an antibody present in the sample to the conjugate.
According to another aspect of the invention there is provided a conjugate or a pharmaceutical composition as described above for use in a method of diagnosis of a microbial infection in a subject.
According to another aspect of the invention there is provided the use of a conjugate or a pharmaceutical composition as described above in a method of making a medicament for use in a method of diagnosis of a microbial infection in a subject.
According to another aspect of the invention there is provided a purified lipid cell-wall component or analog or derivative thereof or a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith for use as a tolerogen to enhance resistance and/or reduce susceptibility to a microbial infection in a subject.
According to another aspect of the invention there is provided the use of a purified lipid cell-wall component or analog or derivative thereof or a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier and purified lipid cell-wall component associated therewith in a method of making a medicament for use as a tolerogen to enhance resistance and/or reduce susceptibility to a microbial infection in a subject.
According to another aspect of the invention there is provided a purified lipid cell-wall component or analog or derivative thereof or a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith for use in a method of enhancing resistance or lowering susceptibility to microbial infections in a subject.
According to another aspect of the invention there is provided use of a purified lipid cell-wall component or analog or derivative thereof or a conjugate or pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith in a method of making a medicament for use in enhancing resistance or lowering susceptibility to microbial infections in a subject.
According to another aspect of the invention there is provided a method of treatment of a microbial infection in a subject comprising the step of administering to the subject a purified bacterial lipid cell-wall component or analog or derivative thereof or a pharmaceutical composition of the invention to the subject.
According to another aspect of the invention there is provided a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith or a purified lipid cell-wall component or analog or derivative thereof for use in a method of treatment of a microbial infection in a subject.
According to another aspect of the invention there is provided the use of a conjugate or pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith or a purified lipid cell-wall component or analog or derivative thereof in a method of making a medicament for use in a method of treatment of a microbial infection in a subject.
The organic carrier may be a protein including bovine serum albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1 molecule, or may be a carbohydrate or may be a micelle.
The method of treatment may be a prophylactic and/or therapeutic method and may be a high zone tolerance treatment or may be a low zone tolerance treatment or a treatment aiming at an idiotypic regulation involving the conjugate or the antibody.
The method may be an immunoregulatory method.
The method of treatment may be a prophylactic method which enhances resistance or reduces susceptibility to a microbial infection in a subject. The prophylactic method may promote an inflammatory response in an infected organ, typically the lungs, kidney and/or liver of the subject. The infected organ is usually the lungs.
According to another aspect of the invention there is provided a method of treatment of an immune disorder in a subject comprising the step of administering to the subject a purified bacterial lipid cell-wall component or analog or derivative thereof or a pharmaceutical composition as described above.
According to another aspect of the invention there is provided a purified lipid cell-wall component or analog or derivative thereof or a conjugate or pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith for use in a method of treatment and/or diagnosis of an immune disorder in a subject.
According to another aspect of the invention there is provided the use of purified lipid cell-wall component or analog or derivative thereof or a conjugate or pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith in a method of making a medicament for use in a method of treatment and/or diagnosis of an autoimmune disease in a subject.
The organic carrier may be a protein including bovine serum albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1 molecule, or may be a carbohydrate or may be a micelle.
According to another aspect of the invention there is provided a purified lipid cell-wall component or analog or derivative thereof or a conjugate or pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith for use in a method of treatment and/or diagnosis of an inflammatory condition in a subject.
According to another aspect of the invention there is provided the use of a purified lipid cell-wall component or analog or derivative thereof or a conjugate or pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith in a method of making a medicament for use in a method of treatment and/or diagnosis of an inflammatory condition in a subject.
The organic carrier may be a protein including bovine serum albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1 molecule, or may be a carbohydrate or may be a micelle.
According to another aspect of the invention there is provided a method of diagnosing an immune disorder in a subject comprising the step of contacting a sample from the subject with a purified lipid cell-wall component or analog or derivative thereof or with a pharmaceutical composition as described above or with a conjugate as described above or with a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith or with a support containing either conjugate; and detecting any reaction between the purified lipid cell-wall component or conjugate and the sample.
More preferably, the method of diagnosis may comprise the step of detecting the binding of an antibody present in the sample to the purified lipid cell-wall component or the conjugate.
The organic carrier may be a protein including bovine serum albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1 molecule, or may be a carbohydrate or may be a micelle.
According to another aspect of the invention there is provided a purified lipid cell-wall component or analog or derivative thereof or a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith for use as a toleragen to enhance resistance and/or reduce susceptibility to an immune disorder or an inflammatory condition in a subject.
According to another aspect of the invention there is provided the use of a purified lipid cell-wall component or analog or derivative thereof or a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith for use in a method of making a medicament as a tolerogen to enhance resistance and/or reduce susceptibility to an immune disorder or an inflammatory condition in a subject.
The organic carrier may be a protein including bovine serum albumin (BSA), gelatins keyhole limpets haemocyanin and the CD1 molecule, or may be a carbohydrate or may be a micelle.
According to another aspect of the invention there is provided a purified lipid cell-wall component or analog or derivative thereof or a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier, except bovine serum albumin (BSA), and a purified lipid cell-wall component associated therewith for use in a method of modulating or manipulating the humoral immune system in a subject for the treatment or prophylaxis of a microbial infection, an immune disorder or an inflammatory condition in a subject.
According to another aspect of the invention there is provided the use of a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier, except bovine serum albumin (BSA), and a purified lipid cell-wall component associated therewith in a method of making a medicament for use in a method of modulating or manipulating the humoral immune system in a subject for the treatment or prophylaxis of a microbial infection, an immune disorder or an inflammatory condition in a subject.
The organic carrier may be a protein including gelatin, keyhole limpets haemocyanin and the CD1 molecule, or may be a carbohydrate or may be a micelle.
According to another aspect of the invention there is provided a purified lipid cell-wall component or analog or derivative thereof or a conjugate or pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith for use in a method of enhancing resistance or lowering susceptibility to inflammatory conditions or allergies in a subject.
According to another aspect of the invention there is provided use of a purified lipid cell-wall component or analog or derivative thereof or a conjugate or pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith in a method of making a medicament for use in enhancing resistance or lowering susceptibility to inflammatory conditions and/or allergies in a subject.
The organic carrier may be a protein including bovine serum albumin (BSA), gelatin, keyhole limpets haemocyanin, the CD1 molecule and a serum protein, or may be a carbohydrate or may be a micelle.
The method of treatment may be a prophylactic and/or therapeutic method and may be a high zone tolerance treatment or may be a low zone tolerance treatment or a treatment aiming at an idiotypic regulation involving the conjugate or the antibody.
The method may be an immunoregulatory method.
The method of treatment may be a prophylactic method which enhances resistance or reduces susceptibility to an immune disorder, inflammatory condition or allergy in a subject. The prophylactic method may suppress inflammation in the joints of the subject.
According to another aspect of the invention there is provided a purified lipid cell-wall component or analog or derivative thereof or a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith for use in the method of modulating or manipulating the cellular immune system in a subject for the treatment or prophylaxis of a microbial infection, an immune disorder or an inflammatory condition in a subject.
According to another aspect of the invention there is provided a purified lipid cell-wall component or analog or derivative thereof or a conjugate or a pharmaceutical composition as described above or a conjugate comprising any organic carrier and a purified lipid cell-wall component associated therewith in a method of making a medicament for use in a method of modulating or manipulating the cellular immune system in a subject for the treatment or prophylaxis of a microbial infection, an immune disorder or an inflammatory condition in a subject.
The organic carrier may be a protein including bovine serum albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1 molecule, or may be a carbohydrate or may be a micelle.
More particularly, the modulation or manipulation of the immune system may be the modulation or manipulation of T-cell effects, such as CD4+, Th0, Th1 and Th2, CD8+or CD4xe2x88x92CD8xe2x88x92(double negative (DN)) T-cells, natural killer (NK) cells, and/or of macrophages.
According to another aspect of the invention there is provided use of a purified lipid cell-wall component in a method of forming a conjugate comprising any organic carrier and the purified lipid cell-wall component, whether the conjugate is produced in vitro or in vivo.
The purified lipid cell-wall component as referred to herein is preferably a bacterial cell-wall component from a bacterium which produces mycolic acids and it may be derived from the genus Mycobacterium, Corynebacterium, Nocardia or Rhodococcus.
The bacterium is preferably Mycobacterium tuberculosis. 
More preferably, the purified lipid cell-wall component is a purified mycolic acid, a mixture of purified mycolic acids or a mycolic acids fraction or derivative originating from a single or different species or a synthetic source. Other lipid cell-wall components may be high molecular weight lipids such as cord factors.
The purified lipid cell-wall component may be a biologically active purified mycolic acid, a mixture of biologically active purified mycolic acids or a biologically active purified mycolic acids fraction or derivative originating from a single or different species or a synthetic source.
The purified lipid cell-wall component may be a resaponified biologically active mycolic acid, a mixture of resaponified biologically active mycolic acids or a biologically active mycolic acids fraction or derivative of resaponified mycolic acids.
The derivative of the purified lipid cell-wall component as referred to herein may be an ester of a mycolic acid, a mixture of esters of mycolic acids or a derivative or fraction thereof.
The microbial infection as referred to herein may be a mycobacterial infection, typically tuberculosis or leprosy.
The immune disorder as referred to herein may be an inflammatory condition. It may be an autoimmune disorder, typically arthritis.
The autoimmune disorder may be of mycobacterial origin.
According to yet another aspect of the invention there is provided a method of separating and purifying a specific microbial cell-wall component of a lipid or carbohydrate nature or a derivative or analog thereof from an extracted mixture of the cell-wall component or derivative or analog thereof and contaminants or from a synthetic mixture of the cell-wall component or derivative or analog thereof and contaminants comprising the steps of:
dissolving the extracted mixture or synthetic mixture in a bi-phasic solvent containing sodium chloride to form a solution;
allowing the solution to separate to form an upper phase and a lower phase;
subjecting the phases to countercurrent distribution/separation comprising a required number of cycles to separate the microbial cell-wall component or analog or derivative thereof in the upper phase or the lower phase; and
removing the separated microbial cell-wall component or derivative or analog thereof from the upper or lower phase.
The method may also comprise the additional pre-purification steps of:
dissolving the extracted mixture of cell-wall component or derivative or analog thereof and contaminants or the synthetic mixture of the cell-wall component or derivative or analog thereof and contaminants in a first solvent without sodium chloride;
adding thereto a second solvent without sodium chloride;
mixing and allowing the solution to separate to form a first upper phase (second solvent) and a first lower phase (first solvent); and
removing the first upper phase and/or the first lower phase for further processing.
Preferably, the lower phase or first lower phase, containing the extracted mixture, is removed and subjected to countercurrent distribution purification, as described above.
The method may also comprise the additional post-purification steps of:
dissolving the extracted microbial cell-wall component or derivative or analog thereof in a suitable solvent; and
adding a precipitant to the solution to precipitate out the dissolved further purified microbial cell-wall component or derivative or analog thereof.
The solvent is preferably chloroform.
The precipitant is preferably acetone.
The method may comprise the steps of:
saponifying a microbial culture prior to preparing therefrom an extracted mixture of a cell-wall component or derivative or analog thereof on which to perform the method of the invention; and
resaponifying the separated and purified microbial cell-wall component or derivative or analog thereof.
According to yet another aspect of the invention a purified mycolic acid or mixture of purified mycolic acids or a fraction thereof is provided in a particular conformation that renders it biologically active.
The purified mycolic acid or mixture of purified mycolic acids may be in a conformation produced by the purified mycolic acid or mixture of purified mycolic acids being in a methyl ester form or a freshly resaponified form.
According to yet another aspect of the invention there is provided a method of preparing detection means for detecting the presence of anti-mycolic acids antibodies, the detection means comprising a solid phase and mycolic acids in a methyl ester form or in a freshly resaponified form associated therewith.
The solid phase may be an ELISA plate.